A monoclonal antibody specific for the red-absorbing form of phytochrome

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.Abbreviations ELISA enzyme-linked immunsorbent assay - kDa kilodalton - mAb monoclonal antibody - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - (A) difference in absorbance (A ₆₆₅ ^Pr –A ₇₃₀ ^Pr )-(A ₆₆₅ ^Pfr –A ₇₃₀ ^Pfr ) - Ar/Afr spectral change ratio (SCR) - max mole fraction of Pfr following saturating red light     

Metabolic switching patterns provide definitive evidence of substrate-specificity and delineate subtle differences in positioning at the aromatase active site [Poster 06]

We found that extensive metabolic switching can be triggered in aromatization. Up to 20–35% of (19-³H₃)-labeled substrate is diverted to non-estrogenic product, namely 1β- and 2β-hydroxy androgens, negating usefulness for [³H]water assays. The strikingly substrate-specific metabolite patterns observed reflect positioning at the active site, and the large kinetic isotope effects involved. This switching is unusual in that it involves: 1) tritium labeling, 2) a multistep enzymic process, and 3) the potential unraveling of an enzymic mechanism.     

Plant Cell Wall Carbohydrates as Substrates for Azospirillum brasiliense

Carbohydrate components (simple sugars and polysaccharides) of cell walls of pearl millet (Pennisetum americanum L., cv. Gahi) were studied as potential substrates for the root-associated diazotroph Azospirillum brasiliense Sp. 7. Simple sugars were utilized, but no evidence was obtained to support the suggestion that the polysaccharide components tested might serve as substrates for growth following hydrolysis by the associated azospirilla.     

Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.     

A reply to Holmes on Gendercide

Warren's book, Gendercide: The Implications of Sex Selection (Totowa, N.J.: Rowman and Allanheld; 1985), was reviewed by Helen Bequaert Holmes in the January 1987 issue of Bioethics. Here, Warren responds to the review by clarifying some of her moral arguments and continuing to defend the point of view that selecting the sex of children before conception or before birth is not always sexist, socially harmful, or disrespectful of the child as an end in itself.     

Growth curves,morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 10⁶ FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×10⁶ FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×10⁶ FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.     

Plasmid profiles and antibiotic susceptibility of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7

Abstract This paper describes the plasmid profiles obtained for 73 of 96 field isolates of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7. We also characterized the antibiotic susceptibilities of these 96 isolates. Because of the high proportion of isolates resistant to some of the antibiotics, no conclusions can be drawn as to the role of plasmids in antibiotic resistance.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency

This paper contains new experimental data on the growth dynamicsof a lobate coastal ctenophore, Mnenmiopsis mccradyi, whichadd significantly to our understanding of the nutritional ecologyof ctenophores and their role as opportunistic predators. Theseexperimental observations were necessary to refine the dynamiccarbon budget presented as a simulation model in another report.The ratio of carbon biomass to dry weight may vary several-folddepending on the nutritional state and size from >12% inwell-fed larvae to <1% in starved adults. Feeding effort(clearance rate) is higher for previously starved animals, fallingsharply within a few hours after re-exposure to food. Directvisual observations of feeding activity of animals confirmedthis. Assimilation efficiency can be high (72%) in these animalsbut if they continue to feed at high food concentrations, incomingfood displaces material which is only partially digested andassimilation efficiency decreases substantially. Except at verylow food concentrations, growth efficiency ranges between 20and 45%. Mnemiopsis, begins to produce eggs at a size much lessthan its maximum. Egg production is very sensitive to food supply,and somatic growth is favored over egg production at low fooddensities. Even though several thousand eggs may be producedover a few days, they represent <2% day^–1 of the carbonbiomass of the ctenophore and several-fold less than respiratorycarbon.